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Characteristics of Cytokines in Regeneration of Injured Peripheral Nerves

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Characteristics of Cytokines in Regeneration of Injured Peripheral Nerves Characteristics of cytokines in regeneration of injured peripheral nerves Ruirui Zhang Nantong University Sailing Chen Nantong University Yinying Shen Nantong University Sheng Yi ( [email protected] ) Nantong University Hui Xu Nantong University Research Keywords: peripheral nerve injury, rat sciatic nerve crush injury, sciatic nerve stumps, dorsal root ganglia, upstream cytokines Posted Date: March 25th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-18898/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on November 23rd, 2020. See the published version at https://doi.org/10.1186/s40779-020-00286-0. Page 1/16 Abstract Background Cytokines are essential cellular modulators of a variety of physiological and pathological activities, including peripheral nerve repair and regeneration. However, the molecular changes of these cellular mediators during peripheral nerve regeneration are not well claried. The study is aimed to discover critical cytokines for the regenerative process of injured peripheral nerves. Methods The sequencing data of the injured nerve stumps and the dorsal root ganglia (DRGs) of Sprague-Dawley (SD) rats subjected to sciatic nerve (SN) crush injury was analyzed to determine expression patterns of genes coding for cytokines. PCR experiments were used to validate the accuracy of sequencing data. Results A total of 46, 52, and 54 upstream cytokines were differentially expressed in SNs at 1 day, 4 days, and 7 days after nerve injury. And a total of 25, 28, and 34 upstream cytokines were differentially expressed in DRGs at these time points. The expression patterns of some essential upstream cytokines were displayed in a heatmap and validated by PCR experiment. Bioinformatic analysis of these differentially expressed upstream cytokines after nerve injury demonstrated that inammatory and immune responses were signicantly involved. Conclusions In summary, the ndings provided an overview of the dynamic changes of cytokines in SNs and DRGs at different time points after rat nerve crush injury, elucidated the biological processes of differentially expressed cytokines, especially the important roles in inammatory and immune responses during peripheral nerve repair and regeneration, and thus might contribute to identication of potential treatments for peripheral nerve repair and regeneration. 1. Introduction Peripheral nerves are vulnerable tissues that are generally defenseless to traumatic injuries caused by bump, stretch, crush, and penetrating wounds and non-traumatic injuries caused by genetic, metabolic, infectious, and medically induced factors (1, 2). Fortunately, unlike nerves in the central nervous system, peripheral nerves can regenerate and achieve certain functional recovery after injury, although fully functional recovery is generally unexpected (3). After peripheral nerve injury, distal nerve stumps undergo Wallerian degeneration, activated Schwann cells and macrophages phagocytosis debris of axon and myelin sheaths, axons of survived neurons regrow toward target tissues for reinnervation (3, 4). Cytokines are a wide category of immunomodulating proteins or peptides including chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors. Cytokines play essential roles in inammation and immune responses and participate in the regulation of the maturation, growth, and responsiveness of a variety of cell populations (5, 6). Cytokines have been identied to be constitutively involved in the nervous system in health and disease (7–10). Cytokines are also extremely critical for peripheral nerve injury and repair as ne-tuned expressions of cytokines modulate the cellular behaviors of Schwann cells, macrophages, and neurons and regulate debris clearance, axon growth, and peripheral nerve regeneration (11). Understanding the molecular changes of these cellular mediators during peripheral nerve regeneration opens new possibilities to improve the repair of injured nerves and to minimize the induction of neuropathic pain (11). On this purpose, previously obtained sequencing data of the injured nerve stumps of Sprague-Dawley (SD) rats subjected to sciatic nerve (SN) crush injury was analyzed to determine expression patterns of genes coding for cytokines (12). Moreover, considering that cytokines retrograde transport to the neuronal bodies and affects neuronal activities, sequencing data of the dorsal root ganglia (DRGs) after rat SN crush injury was also jointly investigated (13). Differentially expressed genes in SNs and DRGs after nerve crush injury were identied and upstream cytokines of these differentially expressed genes were recognized by Ingenuity Pathway Analysis (IPA) bioinformatic tool. Differentially expressed upstream cytokines at 1 day, 4 days, and 7 days after nerve crush injury were subjected Page 2/16 to functional enrichment of Gene Ontology (GO) categories and Kyoto Enrichment of Genes and Genomes (KEGG) pathways according to Database for Annotation, Visualization, and Integrated Discovery (DAVID). 2. Materials And Methods 2.1. Sequencing data RNA deep sequencing data of rat SNs at 0 hour, 1 day, 4 days, 7 days, and 14 days after SN crush injury were conserved in National Center for Biotechnology Information (NCBI) database with the accession number PRJNA394957 (SRP113121). Sequencing data of rat DRGs at 0 hour, 3 hours, 9 hours, 1 day, 4 days, and 7 days after SN crush injury were conserved in NCBI database with the accession number PRJNA547681 (SRP200823). Differentially expressed genes in SNs and DRGs at certain time points after nerve crush injury were selected by comparing their expression levels under the injured status with the expression levels under the uninjured status (0 hour control). Genes with a fold changes < 2 or > -2 and a experimental false discovery rate (FDR) < 0.05 were dened as differentially expressed genes. 2.2. Bioinformatic analysis Upstream cytokines of differentially expressed genes in SNs and DRGs were identied by IPA bioinformatic tool (Ingenuity Systems Inc., Redwood City, CA, USA) for Ingenuity pathway knowledge base (IPKB)-based upstream regulator analysis. Genes coding for cytokines with a fold changes < 2 or > -2 at 1 day, 4 days, or 7 days as compared with 0 hour were dened as differentially expressed cytokines and were subjected to subsequent bioinformatic analyses. Commonly differentially expressed cytokines in SNs and DRGs at 1 day, 4 days, or 7 days after SN crush injury were identied by the Venny 2.1.0 online bioinformatic tool (http://bioinfogp.cnb.csic.es/tools/venny/index.html) (14). The expression proles of these commonly differentially expressed cytokines were demonstrated by a heatmap. Signaling pathways and biological processes involved in differentially expressed upstream cytokines were discovered by DAVID bioinformatic enrichment tools (15, 16). 2.3. Animal surgery and collection of the dorsal root ganglia and SN stumps The conduction of rat SN crush injury and the collection of SNs and DRGs of uninjured and injured rats were performed as previously described (12, 13). Adult male SD rats weighting 180–220 g were obtained from the Experimental Animal Center of Nantong University (Animal licenses No. SCXK [Su] 2014-0001 and SYXK [Su] 2012-0031) and subjected to animal surgery. Rats were anaesthetizated intraperitoneally with a mixture of 85 mg/kg trichloroacetaldehyde monohydrate, 42 mg/kg magnesium sulfate, and 17 mg/kg sodium pentobarbital. SNs at 10 mm above the bifurcation into the tibial and common bular nerves were exposed by a skin incision in the left outer mid-thigh. Exposed SNs were crushed with a forceps at a force of 54 N for 3 times with 10 seconds for each time. Rats underwent SN crush injury were sacriced by decapitation at 1 day, 4 days, and 7 days after animal surgery. Rat underwent sham surgery were sacriced and designated as 0 hour controls. Rat SNs and lumbar 4 to lumbar 6 DRGs were removed for RNA isolation. 2.4. RNA isolation and PCR validation RNA was isolated from rat SNs or lumbar 4 to lumbar 6 DRGs using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Isolated RNA samples were reverse transcribed to cDNA using the Prime-Script reagent kit (TaKaRa, Dalian, Liaoning, China) and subjected to PCR experiments using an Applied Biosystems Stepone System (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq (TaKaRa) and specic primer pairs of target genes chemokine (C-X-C motif) ligand 10 (Cxcl10) and interleukin 1 receptor antagonist (Il1rn) and reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of primer pairs were as follows: Cxcl10 (forward) 5’-GAAGCACCATGAACCCAAGT-3’ and Cxcl10 (reverse) 5’-CAACATGCGGACAGGATAGA-3’; Il1rn (forward) 5’-CTTACCTTCATCCGCTCCGA-3’ and Il1rn (reverse) 5’-GATCAGGCAGTTGGTGGTCAT-3’; and GAPDH (forward) 5’- ACAGCAACAGGGTGGTGGAC-3’ and GAPDH (reverse) 5’-TTTGAGGGTGCAGCGAACTT-3’. Relative mRNA abundances of Cxcl10 −ΔΔCt and Il1rn were determined using the comparative 2 method, in which ΔCt = Ct(injured)-Ct(uninjured) and ΔΔCt = Ct(target gene)- Ct(reference gene) (17). Page 3/16 2.5. Statistical analysis Summarized PCR results were reported as means ± SEM with n = 3. Statistical analysis and graphs were generated using GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA). The p-value was determined from one-way analysis of variance (ANOVA) and a p-value < 0.05 was considered as statistically signicant. 3. Results
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